This proposal focuses on Bacillus anthracis virulence gene control, with emphasis on the expression and function of the major virulence gene regulator AtxA. B. anthracis is a developmental bacterium existing in two distinct physiological states, metabolically active vegetative cells and dormant spores. The spore - vegetative cell cycle is of fundamental importance in pathogenesis. Spores enter a mammalian host and germinate to become vegetative cells. During infection, B. anthracis remains vegetative and synthesizes capsule, anthrax toxin proteins, and other factors that facilitate pathogenesis; sporulation does not occur. However, upon death of the host when vegetative cells are exposed to the environment, toxins and capsule are not produced and B. anthracis sporulates efficiently. Thus, the inverse relationship between virulence factor synthesis and sporulation is physiologically significant for anthrax pathogenesis. The major link between these two processes is AtxA. Investigations in our laboratory and others have revealed a relationship between this major pleiotropic regulator of virulence gene expression and the physiological state of cells grown in batch culture. B. anthracis homologues of developmental regulators that have been well-characterized in the non- pathogenic Bacillus species B. subtilis have been linked to transcription of atxA. Moreover, AtxA function appears to be controlled by post-translational modification and host-associated signals. In this work, we will (1) perform functional analyses of AtxA, (2) determine molecular mechanisms for control of atxA expression, and (3) establish the physiological relevance of atxA regulators and specific functional modifications of the AtxA protein in anthrax disease. B. anthracis is a Category A Select Agent and the development of more effective chemotherapeutics, vaccines, and diagnostics for anthrax disease is a national priority. Defining and characterizing the molecular mechanisms by which B. anthracis controls virulence gene expression will advance our fundamental understanding of B. anthracis pathogenesis and facilitate a rational approach for the development of anthrax countermeasures. Furthermore, information regarding the molecular function of the novel regulator AtxA will contribute to our overall understanding of mechanisms of gene regulation. Finally, information obtained in this study can be applied to other infectious agents because B. anthracis is an ideal model for multiple features of host-pathogen interactions, environmental signaling in bacteria, and other aspects of bacterial physiology. PUBLIC HEALTH RELEVANCE: B. anthracis is a Category A Select Agent and the development of more effective chemotherapeutics, vaccines, and diagnostics for anthrax disease is a national priority. Defining and characterizing the molecular mechanisms by which B. anthracis controls virulence gene expression will advance our fundamental understanding of B. anthracis pathogenesis and facilitate a rational approach for the development of anthrax countermeasures. Exploration of the molecular basis for toxin and capsule gene expression in B. anthracis in combination with animal studies assessing significance of key regulators will fill a critical gap in knowledge regarding the spatial and temporal nature of B. anthracis development and virulence factor synthesis within the host. Furthermore, information obtained in this study can be applied to other infectious agents because B. anthracis is an ideal model for multiple features of host-pathogen interactions, environmental signaling in bacteria, and other aspects of bacterial physiology.